Hairpin and duplex formation of the DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) in solution. An NMR study.

The partly self-complementary DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) was investigated by NMR spectroscopy in solution. It is demonstrated that this peculiar DNA fragment, under suitable conditions of concentration, salt and temperature, exclusively prefers to adopt a monomeric hairpin form with a stem of three Watson-Crick type base pairs and a loop of two residues. At high single strand concentration (8 mM DNA) and low temperature (i.e. below 295 K) the hairpin occurs in slow equilibrium with a B-dimer structure. At high ionic strength (greater than or equal to 100 mM Na+) and/or in the presence of methanol a third species appears, which is assigned to a Z-like dimer. In the B form, as well as in the Z dimer, the two central base pairs form G.T wobble pairs with the bases as major tautomers.     

An NMR study of polymorphous behaviour of the mismatched DNA octamer d(m5C-G-m5C-G-A-G-m5C-G) in solution. The B-duplex and hairpin forms

By means of one- and two-dimensional NMR spectroscopy the solution structures of the partly self-complementary octamer d(m5C-G-m5C-G-A-G-m5C-G) were investigated. It is shown that this DNA fragment, under conditions of high DNA concentration (8 mM DNA) and/or high ionic strength prefers to adopt a duplex structure. At low DNA concentration (0.4 mM DNA), the duplex exists in a 1:1 slow equilibrium with a monomeric hairpin form. Addition of salt destabilizes the hairpin structure in favour of the dimer. At high temperatures the hairpin form, as well as the dimer structure, exist in a fast equilibrium with the random-coil form. For the hairpin/random-coil equilibrium a Tm of 329 K and a delta H degree of -121 kJ.mol-1 were deduced. These thermodynamic parameters are independent of the DNA concentration, as is expected for a monomeric structure. For the dimer to coil transition a Tm of 359 K (1 M DNA) and a delta H degree of -285 kJ.mol duplex-1 were derived. The thermodynamic data of the hairpin-coil transition mutually agree with those recently reported for the hairpin to random coil equilibrium of the DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) [Orbons, L. P. M., van der Marel, G. A., van Boom, J. H. & Altona, C. (1987) J. Biomol. Struct. Dyns. 4, 939-963]. It is demonstrated that the dimer structure exhibits B-DNA characteristics, as is witnessed by the NOESY experiments and the analysis of the proton-proton coupling data. It is shown that the base-pair formation of the G x A mismatches is anti-anti. A comparison of 1H and 31P chemical-shift data of the title compound with those of a well-characterized B-DNA structure reveals large differences in the dm5C(3)-dG(4)-dA(5) part of the mismatched dimer structure. These differences apparently indicate some major local structural changes due to the incorporation of the G x A mismatches. Under the most extreme conditions used (i.e. up to 3 M NaCl or 75% CH3OH in the presence of 10 mM MgCl2) no Z-DNA structure was observed. It is shown that the structural features of the hairpin form of the title compound mimic those of the hairpin structure of d(m5C-G-m5C-G-T-G-m5C-G). An energy-minimized model of the hairpin form is given.     

Conformational analysis of the B and Z forms of the d(m5C-G)3 and d(br5C-G)3 hexamers in solution. A 300-MHz and 500-MHz NMR study

The B and the Z forms of the DNA hexamers d(m5C-G)3 and d(br5C-G)3 were investigated by means of NMR spectroscopy. It is demonstrated that the low-salt form of d(m5C-G)3 is a B DNA structure. The form, which becomes increasingly predominant when increasing amounts of MgCl2 and/or methanol are added to the solution, has Z DNA characteristics. It is shown that the major geometrical features of the Z form of d(m5C-G)3 in the crystal structure are maintained in solution, with the dC residues S sugar conformation, gamma + and the base in the anti orientation and the dG residues N (except the 3'-terminal residue), gamma t and syn. Neither the Z form of the methylated nor that of the brominated compound resembles the Z' form, in which the deoxy guanosine sugar rings adopt a C1'-exo conformation. Substitution of m5C by br5C causes no perceptible conformational changes in either the B or in the Z forms.     

Influence of dA X dT and d(2aminoA) X dT base pairs on the B in equilibrium Z transition of DNA fragments. 1H-NMR study of d(C-G-C-A-m5C-G-T-G-m5C-G), d(m5C-G-C-A-m5C-G-T-G-C-G) and d(C-2aminoA-C-G-T-G)

The Helical structures of d(C-G-C-A-m5C-G-T-G-m5C-G), d(m5C-G-C-A-m5C-G-T-G-C-G) and d(C-2aminoA-C-G-T-G) were studied in aqueous solution at various salt concentrations and temperatures by 1H-NMR spectroscopy. In 0.1 M NaCl solution only the B form was evidenced for these DNA fragments whereas in 4 M NaCl both B and Z forms, in slow exchange on the NMR time scale, were observed. Under these conditions the Z form accounted for less than 60% of the decamer conformation; conversely d(C-G)3 hexamers containing methylated cytidines were predominantly in the Z form (greater than 90%) [Tran-Dinh et al. (1984) Biochemistry 23, 1362; Cavaillès et al. (1984) J. Biomol. Struct. Dyn. 1, 1347-1371]. On the other hand, d(C-2aminoA-C-G-T-G) in which the d(2aminoA) X dT base pair forms three hydrogen bonds, was found to adopt the Z conformation in 4M NaCl solution which was not the case for d(C-A-C-G-T-G) (unpublished results). The present study shows that the B in equilibrium Z transition in solution is highly sequence-dependent and that correlation exists between the stability of the duplexes (essentially governed by the number of hydrogen bonds between complementary bases) and their ability to adopt the Z conformation.     

An NMR Study of the Polymorphous Behavior of the Mismatched DNA Octamer d(m5C-G-m5C-G-T-G-m5C-G) in Solution. The B,Z, and Hairpin Forms

Abstract

The polymorphism exhibited by the mismatched octamer d(m ⁵C-G-m⁵C-G-T-G-m⁵C-G), as a function of the temperature, DNA concentration and ionic strength, was investigated by means of NMR spectroscopy.

It is shown that this partly self-complementary DNA fragment, under conditions of low DNA concentration (0.4 mM) and low ionic strength, exclusively prefers to adopt a monomeric hairpin form, which consists of a stem of three Watson-Crick-type base pairs and a loop of only two residues. This in striking contrast with earlier intimations in literature, which postulated that in oligonucleotides loop formations containing only two residues are sterically impossible. Moreover, the hairpin form displays an unusual stability in comparison with previously reported hairpins. AT_m of 332 K and a ΔH° of—130 kj · mol^?1 were calculated for the hairpin to random coil transition.

At high DNA concentration (8 mM)and/or upon the addition of sodium chloride the hairpin form occurs in slow exchange with a B-DNA dimer structure (approximately 20% at 270 K, no added salt), which comprises two central GxT-mismatched base pairs with the bases as major tautomers.

At higher ionic strength (> 100 mM NaCI), or upon the addition of methanol, a third species appears, which is in slow exchange with both the B dimer and the hairpin form. This third species could be identified with a Z DNA form, comprising two GxT mismatches with the bases as major tautomers, with the guanine bases syn and the cytosine and thymine bases anti.     

The B and Z forms of the d(m5C-G)3 and d(br5C-G)3 hexamers in solution. A 300-MHz and 500-MHz two-dimensional NMR study

The non-exchangeable proton resonances of the hexadeoxynucleoside pentakisphosphates d(m5C-G)3 and d(br5C-G)3 in the B form as well as in the Z form were assigned by means of two-dimensional correlated spectroscopy and two-dimensional nuclear Overhauser enhancement spectroscopy. The complete proton NMR spectrum of the B form of the methylated compound was assigned in a pure 2H2O solution as well as in a 2H2O/C2H3O2H mixed solvent, containing 5 mM MgCl2. In the latter solvent the B form occurs in slow equilibrium (on the NMR time scale) with the Z form, the resonances of which also were fully assigned. The proton resonances of the B and Z forms of the brominated fragment were assigned in a 2H2O/C2H3O2H solution containing 5 mM MgCl2. A new and general method is described for the sequential assignment of the non-exchangeable proton resonances of oligonucleotides in the Z form.     

Conformational and model-building studies of the hairpin form of the mismatched DNA octamer d(m5C-G-m5C-G-T-G-m5C-G)

The hairpin form of the mismatched octamer d(m5C-G-m5C-G-T-G-m5C-G) was studied by means of NMR spectroscopy. In a companion study it is shown that the hairpin form of this DNA fragment consists of a structure with a stem of three Watson-Crick-type base pairs and a loop consisting of only two nucleotides. The non-exchangeable proton resonances were assigned by means of two-dimensional correlation spectroscopy and two-dimensional nuclear Overhauser effect spectroscopy. Proton-proton coupling constants were used for the conformational analysis of the deoxyribose ring and for some of the backbone torsion angles. From the two-dimensional NMR spectra and the coupling-constant analysis it is concluded that: (i) the stem of the hairpin exhibits B-DNA characteristics; (ii) the sugar rings are not conformationally pure, but display a certain amount of conformational flexibility; (iii) the stacking interaction in the stem of the hairpin is elongated from the 3'-side in a more or less regular fashion with the two loop nucleotides; (iv) at the 5'-side of the stem a stacking discontinuity occurs between the stem and the loop; (v) at the 5'-side of the stem the loop is closed by means of a sharp backbone turn which involves unusual gamma' and beta+ torsion angles in residue dG(6). The NMR results led to the construction of a hairpin-loop model which was energy-minimized by means of a molecular-mechanics program. The results clearly show that a DNA hairpin-loop structure in which the loop consists of only two nucleotides bridging the minor groove in a straightforward fashion, (i) causes no undue steric strain, and (ii) involves well-known conformational principles throughout the course of the backbone. The hairpin form of the title compound is compared with the hairpin form of d(A-T-C-C-T-A-T4-T-A-G-G-A-T), in which the central -T4- part forms a loop of four nucleotides. Both models display similarities as far as stacking interactions are concerned.     

Two-dimensional 1H NMR study of d(br5C-G)3 in the Z-form. Self association and flexibility of the left-handed double helix

The Z conformation of the auto complementary hexanucleoside pentaphosphate d(br5C-G)3 in 1 M NaClO4 solution has been investigated by using 2D NMR techniques. NOESY experiments performed at different temperatures show that the oligonucleotide exhibits end-to-end associations at room temperature. The conformation of the hexanucleotide molecules is very similar to that found in the crystal which was described by Chevrier et al. (J. Mol. Biol., 1986, 188, 707-719) as a Z-I form. When the temperature is increased the aggregates are dissociated and a conformational change is observed which is interpreted as a Z-I in equilibrium Z-II transition.     

Conformational and Model-Building Studies of the Hairpin Form of the Mismatched DNA Octamer d(m5C-G-m5C-G-T-G-m5C-G)

Abstract

The hairpin form of the mismatched octamer d(m⁵C-G-m⁵C-G-T-G-m⁵C-G) was studied by means of NMR spectroscopy. In a companion study it is shown that the hairpin form of this DNA fragment consists of a structure with a stem of three Watson-Crick-type base pairs and a loop consisting of only two nucleotides. The non-exchangeable proton resonances were assigned by means of two-dimensional correlation spectroscopy and two-dimensional nuclear Overhauser effect spectroscopy. Proton-proton coupling constants were used for the conformational analysis of the deoxyribose ring and for some of the backbone torsion angles. From the two-dimensional NMR spectra and the coupling-constant analysis it is concluded that: (i) the stem of the hairpin exhibits B-DNA characteristics; (ii) the sugar rings are not conformationally pure, but display a certain amount of conformational flexibility; (iii) the stacking interaction in the stem of the hairpin is elongated from the 3′-side in a more or less regular fashion with the two loop nucleotides; (iv) at the 5′-side of the stem a stacking discontinuity occurs between the stem and the loop; (v) at the 5′-side of the stem the loop is closed by means of a sharp backbone turn which involves unusual γ^t and β⁺ torsion angles in residue dG(6).

The NMR results led to the construction of a hairpin-loop model which was energy-minimized by means of a molecular-mechanics program. The results clearly show that a DNA hairpin-loop structure in which the loop consists of only two nucleotides bridging the minor groove in a straightforward fashion, (i) causes no undue steric strain, and (ii) involves well-known conformational principles throughout the course of the backbone.

The hairpin form of the title compound is compared with the hairpin form of d(A-T-C-C-T- A-T₄-T-A-G-G-A-T), in which the central -T₄- part forms a loop of four nucleotides. Both models display similarities as far as stacking interactions are concerned.     

Z helix-coil transition of d(C-Br8G-C-G-C-Br8G) studied by CD, 1H-NMR and IR spectroscopies

The conformation of d(C-Br8G-C-G-C-Br8G) in aqueous solution was studied by CD and 1H-NMR spectroscopy and in condensed phase by IR spectroscopy. Whether in 0.1 M or 3 M NaCl solution or in film the only double helical structure adopted by brominated d(C-G)3 oligomer is the Z form. The IR spectrum of the film presents all the characteristic absorptions of the Z conformation and in particular is indicative of a syn conformation for the central guanosine as well as for the brominated one. Imino proton resonances of d(C-Br8G-C-G-C-Br8G) demonstrating the duplex formation were observed up to 60 degrees C. It is interesting to note that the significant highfield shifts of the dC H5" exocyclic sugar protons characteristic of the non exchangeable proton spectra of d(C-G)3 containing 5-methyl dC residues in the Z form were also detected in the proton spectrum of brominated oligomer. Whereas formation of the Z helix of methylated d(C-G)3 oligomers dependent on the salt concentration was found to occur via the preliminary formation of a B helix even in 4 M NaCl solution, the Z helix of d(C-Br8G-C-G-C-Br8G) is obtained directly from the coil form. However, IR data suggest that in the Z form of d(C-Br8G-C-G-C-Br8G), the overlapping of the base planes should be slightly different in comparison with the stacking observed in d(C-G)3 crystals. The kinetic data (activation energy and lifetime) of the Z helix-coil transition of brominated d(C-G)3 are compared to those of the B helix-coil transition observed for methylated d(C-G)3 in 0.1 M NaCl solution while the thermodynamic data of these two reactions (enthalpy and midpoint temperature) are slightly different.     

Multivalent ions are necessary for poly[d(AC).d(GT)] to assume the Z form: a CD study.

In previous work, it was shown that poly [d(AC) · d(GT)] could be forced into the Z form by strong dehydrating conditions, provided EDTA was not present. Presumably multivalent impurities were also necessary for the transition. In order to gain control over the B to Z transition for this DNA, we carefully removed all divalent contaminants from the sample and asked the obvious question: What ions are necessary for the transition under dehydrating conditions? We systematically investigated the effect of various multivalent ions. The common contaminants Ca²⁺, Mg²⁺, and Fe³⁺ will not cause the transition, but Co²⁺ and Ni²⁺ facilitate the transition, undoubtedly because of their well-known propensity to bind to purine N7. Since the transition also depends on the synergistic dehydrating action of sodium perchlorate and ethanol, we include CD spectra for the independent variations of these two factors. In addition, vacuum-uv CD spectra for the A form and various B forms of poly [d (AC) · d (GT)] are presented for the first time.     

Conformational flexibility in the right-handed and in the left-handed double helices of Na+-d(m5C-G-T-A-m5C-G) studied by IR spectroscopy.

The X ray diffraction study of a d(m5C-G-A-T-m5C-G) crystals has shown the existence of a 2 conformation while the Raman spectroscopy study of the same fragment in solution showed that then the oligonucleotide adopted a B geometry. Infrared spectroscopy has allowed us to study this sequence in films in a wide range of hydrations and to vary the water content of the sample at different rates. We have thus obtained four I.R. spectra, of the B and 2 conformations respectively at 100% and 98% relative humidities, of another 2 form with a different geometry of the phosphate groups at relative humidities below 98% and in addition a fourth spectrum recorded after a rapid dehydration of the sample which is then blocked in a right-handed form at low R.H. In this case the structure of the phosphodiester chain may be not uniform. The results are discussed by comparison with previously obtained I.R. spectra of poly d(G-C), poly d(A-C). d(G-T), d'm5C-G-A-m5C-G-T-G-C-G) d(m5C-G-C-G-m5C-G) and d(C-G-m5C-G).     

1H NMR and circular dichroism studies of the B and Z conformations of the self-complementary deoxyhexanucleotide d(m5C-G-C-G-m5-C-G): mechanism of the Z-B-coil transitions

The double-helical conformations of d(m5-C-G-C-G-m5-C-G) in aqueous solution were studied by circular dichroism and 1H NMR spectroscopy. In 0.1 M NaCl, only the B form is detected whereas the Z form is strongly predominant in 3 M NaCl. In the presence of 2 M NaCl, two resonance signals corresponding to the B and Z duplexes were observed for each proton below 50 degrees C, indicating a slow exchange between B and Z. However, the B-Z exchange becomes intermediate or fast in the 55-80 degrees C temperature interval. By contrast the exchange between B helix and single-stranded (or coil) forms is much faster for the same temperature conditions. The Z form is only detectable when the coil form is practically absent. With decreasing temperature the B form decreases in favor of the Z form. From proton line-width measurements under various experimental conditions, it was also shown that Z exchanges only with B, while the latter also exchanges with the single-stranded form (S): Z in equilibrium B in equilibrium S. The enthalpy value is about 8 +/- 1 kcal/mol for the B-Z transition and about 40 +/- 2 kcal/mol for the B-S dissociation (2 M NaCl solution). The activation energy is about 47 +/- 2 kcal/mol for the Z----B and 39 +/- 2 kcal/mol for the B----Z reaction. Very good agreement between the experimental results and computed data (based on the above kinetic reaction model) was found for the B, Z, and coil proportions. The B-Z transition of methylated d(C-G)n oligomers is only possible when the Watson-Crick hydrogen bonds between the CG base pairs are firmly maintained; otherwise, the transformation from B to Z would not occur, and B-S dissociation would take place instead.     

Conformational lability of poly(dG-m5dC):poly(dG-m5dC).

The remarkable conformational lability of poly(dG-m5dC):poly(dG-m5dC) is demonstrated by the observation of an acid-mediated conformational hysteresis. An acid-mediated Z conformation that exists in solutions containing low sodium concentrations that would normally favor the B conformation is described in this report. This Z conformation is reached by an acid-base titration of a B-poly(dG-m5dC):poly(dG-m5dC) solution which is not far from the B-Z transition midpoint. The resulting Z conformation is thermally very stable, with direct melting into single strands at approximately 100 degrees C. In contrast, the B form DNA, initially in solutions of the same ionic strength but without exposure to acidic pH, exhibits a biphasic melting profile, with conversion into the Z form (with high cooperativity) prior to an eventual denaturation into single strands at around 100 degrees C. Cooling experiments reveal that such biphasic transitions are quite reversible. The transition midpoint for the thermally poised B to Z transformation depends strongly on the NaCl concentration and varies with sample batch. The acid-mediated Z form binds ethidium more weakly than its B counterpart, and the ethidium induced Z to B conversion occurs in a step-wise (non-allosteric) fashion without the requirement of a threshold concentration. The acid-mediated as well as the thermally poised Z conformations are reversed by the addition of EDTA, suggesting the involvement of trace amounts of multivalent metal ions.     

B,Z conformations and mechanism of the Z-B-coil transitions of the self-complementary deoxy-hexanucleotide d(C-G-m5C-G-C-G) by 1H-NMR and CD spectroscopy

The helical structures of d(C-G-m5C-G-C-G) were studied in aqueous solution at various salt concentrations and temperatures by CD and 1H-NMR spectroscopy. At room temperature only the B form is observed in 0.1 M NaCl whereas the B and Z forms are simultaneously present in 1.8 M NaCl. At high salt concentration (4 M NaCl) the Z form is largely predominant (greater than 95%). The Z form proton resonances were assigned by using the polarisation transfer method (between B and Z at 1.8 M NaCl) and by proton-proton decoupling (at high salt concentration). The Z-B-Coil transitions were studied as a function of temperature with the 1.8 M NaCl solution. At high temperature (95 degrees C) only the coil form (S) is present. Below 55 degrees C the coil proportion is negligible, and the B-Z exchange is slow. The disappearance of the coil gives rise at first to the B form and on lowering the temperature the Z proportion increases to the detriment of the B form. Proton linewidth, relaxation and polarisation transfer studies confirm the conclusion in the previous report on d(m5C-G-C-G-m5C-G) (Tran-Dinh et al Biochemistry 1984 in the press) that Z exchanges only with B whereas the latter also exchanges with S,Z in equilibrium B in equilibrium S. The present data show that even at high salt concentration where only the Z form of d(C-G-m5C-G-C-G) is observed the Z-S transition also passes through the B form as an intermediate stage. The B-Z transition takes place when the Watson-Crick hydrogen bonds are firmly maintained and is greatly favoured when there are three hydrogen bonds between the base-pairs.     

Z Helix-coil Transition of d(C-Br8G-C-G-C-Br8G) Studied by CD, 1H-NMR and IR Spectroscopies

Abstract

The conformation of díC-Bi⁸G-C-G-C-Br⁸G) in aqueous solution was studied by CD and ¹H-NMR spectroscopy and in condensed phase by IR spectroscopy. Whether in 0.1 M or 3 M NaCl solution or in film the only double helical structure adopted by brominated d(C-G)₃ oligomer is the Z form. The IR spectrum of the film presents all the characteristic absorptions of the Z conformation and in particular is indicative of a syn conformation for the central guanosine as well as for the brominated one. Imino proton resonances of diC-Bi⁸G-C- G-C-Br⁸G) demonstrating the duplex formation were observed up to 60°C. It is interesting to note that the significant highfield shifts of the dC H₅″ exocyclic sugar protons characteristic of the non exchangeable proton spectra of d(C-G)₃ containing 5-methyl dC residues in the Z form were also detected in the proton spectrum of brominated oligomer. Whereas formation of the Z helix of methylated d(C-G)₃ oligomers dependent on the salt concentration was found to occur via the preliminary formation of a B helix even in 4 M NaCl solution, the Z helix of d(C-Br⁸G-C-G-C-Br⁸G) is obtained directly from the coil form. However, IR data suggest that in the Z form of dlC-Bi⁸G-C-G-C-Bi⁸G), the overlapping of the base planes should be slightly different in comparison with the stacking observed in d(C-G)₃ crystals. The kinetic data (activation energy and lifetime) of the Z helix-coil transition of brominated d(C-G)₃ are compared to those of the B helix-coil transition observed for methylated d(C-G)₃ in 0.1 M NaCl solution while the thermodynamic data of these two reactions (enthalpy and midpoint temperature) are slightly different.     

B,Z Conformations and Mechanism of the Z-B-Coil Transitions of the Self-Complementary Deoxy-hexanucleotide d(C-G-m5C-G-C-G) by 1H-NMR and CD Spectroscopy

Abstract

The helical structures of d(C-G-m⁵C-G-C-G) were studied in aqueous solution at various salt concentrations and temperatures by CD and ¹H-NMR spectroscopy. At room temperature only the B form is observed in 0.1 M NaCl whereas the B and Z forms are simultaneously present in 1.8 M NaCl. At high salt concentration (4 M NaCl) the Z form is largely predominant (> 95%). The Z form proton resonances were assigned by using the polarisation transfer method (between B and Z at 1.8 M NaCl) and by proton-proton decoupling (at high salt concentration).

The Z-B-Coil transitions were studied as a function of temperature with the 1.8 M NaCl solution. At high temperature (95°C) only the coil form (S) is present. Below 55°C the coil proportion is negligible, and the B-Z exchange is slow. The disappearance of the coil gives rise at first to the B form and on lowering the temperature the Z proportion increases to the detriment of the B form. Proton linewidth, relaxation and polarisation transfer studies confirm the conclusion in the previous report on d(m⁵C-G-C-G-m⁵C-G) (Tran-Dinh et al Biochemistry 1984 in the press) that Z exchanges only with B whereas the latter also exchanges with S,Z ? B ? S. The present data show that even at high salt concentration where only the Z form of d(C-G-m⁵C-G-C-G) is observed the Z-S transition also passes through the B form as an intermediate stage. The B-Z transition takes place when the Watson-Crick hydrogen bonds are firmly maintained and is greatly favoured when there are three hydrogen bonds between the base-pairs.     

Thermal stability of the Z-conformation of the tetranucleoside triphosphate (m5dC-dG)2

The tetranucleoside triphosphate d(m5C-G)2 has been studied in solution by circular dichroism and 31P nuclear magnetic resonance as a function of temperature, in presence of 3 M NaClO4. It is shown that in such high ionic strength d(m5C-G)2 may adopt a Z-like conformation for temperatures lower than 5 degrees C. At these temperatures, another conformation, in slow equilibrium with the Z-like one, is also detected. Increasing the temperature leads to a transition from the Z-like conformation to intermediate forms before melting. It is demonstrated that these intermediates are not the B form.     

Thermodynamic behaviour of the heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG) forming B and Z hairpins in aqueous solution.

UV and CD data of the partially self-complementary heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG), obtained as a function of temperature, salt and strand concentration, show that: at low NaCl and strand concentration the oligomer exhibits, on increasing the temperature, a biphasic thermal profile which is indicative of two structural transitions, from dimeric duplex to hairpin and from hairpin to coil; the loop stabilizes enthalpically both B and Z hairpin structures with respect to the corresponding unconstrained hexamer d(CGCGCG) by a few Kcal/mol; the oligomer undergoes a B-Z transition which appears to be complete, at 0 degree C, when induced by NaClO4; by contrast the B-Z transition induced by NaCl does not reach completeness even at salt saturation. The independence of the denaturation temperature, at high salt conditions, on the oligomer concentration indicates that the Z structure is present also in the hairpin.     

Thermal stability, structural features, and B-to-Z transition in DNA tetraloop hairpins as determined by optical spectroscopy in d(CG)(3)T(4)(CG)(3) and d(CG)(3)A(4)(CG)(3) oligodeoxynucleotides

NMR and CD data have previously shown the formation of the T(4) tetraloop hairpin in aqueous solutions, as well as the possibility of the B-to-Z transition in its stem in high salt concentration conditions. It has been shown that the stem B-to-Z transition in T(4) hairpins leads to S (south)- to N (north)-type conformational changes in the loop sugars, as well as anti to syn orientations in the loop bases. In this article, we have compared by means of UV absorption, CD, Raman, and Fourier transform infrared (FTIR), the thermodynamic and structural properties of the T(4) and A(4) tetraloop hairpins formed in 5'-d(CGCGCG-TTTT-CGCGCG)-3' and 5'-d(CGCGCG-AAAA-CGCGCG)-3', respectively. In presence of 5M NaClO(4), a complete B-to-Z transition of the stems is first proved by CD spectra. UV melting profiles are consistent with a higher thermal stability of the T(4) hairpin compared to the A(4) hairpin. Order-to-disorder transition of both hairpins has also been analyzed by means of Raman spectra recorded as a function of temperature. A clear Z-to-B transition of the stem has been confirmed in the T(4) hairpin, and not in the A(4) hairpin. With a right-handed stem, Raman and FTIR spectra have confirmed the C2'-endo/anti conformation for all the T(4) loop nucleosides. With a left-handed stem, a part of the T(4) loop sugars adopt a N-type (C3'-endo) conformation, and the C3'-endo/syn conformation seems to be the preferred one for the dA residues involved in the A(4) tetraloop.